Abstract The long-term product goal of this project is a small molecule rheumatoid arthritis (RA) therapeutic which acts by reducing the inflammatory response triggered by the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF). The therapeutic benefit of MIF inhibition in RA disease models has been well established by small molecules that bind to a catalytic site of the MIF trimer that mediates the cytokine's vestigial (non-physiological) tautomerase activity. However, allosteric binding of inhibitors in regions outside the tautomerase pocket to date remains ill investigated as a therapeutic approach to blocking MIF's cytokine activities. Our research project focuses on therapeutic development of such allosteric MIF inhibitors. Through structure-based drug design, we have obtained a unique class of compounds, which as revealed by crystallography, bind on the surface of the MIF trimer directly above the tautomerase pocket and overlap the MIF's CD74 receptor binding site that is central to MIF function. In vitro tautomerase, CD74-binding, and bioassays revealed that these allosteric inhibitors not only blocked the activities of MIF, but also those of D- dopachrome tautomerase (D-DT or MIF-2), the MIF homolog in humans whose simultaneous inhibition in MIF- related diseases appears necessary for therapeutic benefit. These preliminary results support further development of this class of allosteric MIF inhibitors as leads for MIF-directed RA therapy. Our hypothesis is that this class of MIF/D-DT allosteric inhibitors will reduce the inflammatory responses triggered by these cytokines, and therefore will prove beneficial in treating RA. In this project, building from our extensive preliminary data, we propose to use medicinal chemistry guided by structural studies to modify the inhibitors for improved MIF and D-DT-inhibition in an effort to obtain molecules that are efficacious in the RA mouse model. The work proposed in the three specific aims of our project focuses on (1) modifying the inhibitors to obtain a structure-activity relationship, (2) introducing functional groups to gradually improve their target binding and potency in MIF-mediated tautomerase and bioassays and (3) evaluating efficacy in the mouse model of collagen-induced arthritis. These efforts are expected to yield a lead compound suitable for further development towards an orally bio-available small molecule MIF-directed therapeutic for RA.